This application claims the benefit of Korean Patent Application No. 10-2004-0088907, filed on Nov. 3, 2004, in the Korean Intellectual Property Office, the disclosure of which is incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention relates to an apparatus and method for purification of nucleic acids by phase separation using a laser and beads.
2. Description of the Related Art
An efficient extraction of DNA from cells is necessary for many applications and is essential for molecular diagnostics, specifically for pathogen identification and quantification. Molecular diagnostics is generally performed by DNA amplification after DNA extraction steps. DNA amplification reactions include polymerase chain reaction (PCR), ligase chain reaction, stranded-displacement amplification, nucleic acid-based amplification, repair chain reaction, helicase chain reaction, QB replicase amplification, ligation activated transcription.
Isolation methods of DNA from cells were provided using materials that have the proclivity of binding DNA. Example materials of isolation methods of DNA are silica, glass fiber, anion exchange resins and magnetic beads (Rudi, K. et al., Biotechniqures 22, 506-511 (1997); and Deggerdal, A. et al., Biotechniqures 22, 554-557 (1997)). To avoid the manual steps and to remove operator error, several automatic machines were developed for high-throughput DNA extraction.
Cell lysis is conventionally performed by mechanical, chemical, thermal, electrical, ultrasonic and microwave method (Michael T. Taylor et al., Anal.Chem., 73, 492-496 (2001)).
Laser has many advantages for disruption of cells and highly applicable to Lab-On-a-Chip (LOC) (Huaina Li et al., Anal Chem, 73, 4625-4631 (2001)).
U.S. Patent Publication No. 2003/96429 A1 discloses a laser-induced cell lysis system. When only a laser is used, an efficient cell lysis does not occur. As a result of performing an experiment using E. coli placed in a very clear solution, it is confirmed that when irradiating only a laser, a low cell lysis efficiency is obtained. A concentration of DNA measured after irradiating a laser for 150 seconds is 3.77 ng/μl because the laser energy is not efficiently transferred to the cells. A concentration of DNA measured after boiling cells at 95° C. for 5 minutes by means of a conventional heating method is 6.15 ng/μl.
U.S. Pat. No. 6,685,730 discloses optically-absorbing nanoparticles for enhanced tissue repair. This patent includes a method of joining tissue comprising: delivering nanoparticles having dimensions of from 1 to 1000 nanometers that absorb light at one or more wavelengths to the tissue to be joined; and exposing said nanoparticles to light at one or more wavelengths that are absorbed by the nanoparticles. This method causes only a loss of function of cells by using a laser and nanoparticles and there is no description of a method of disrupting cells by vibrating a solution containing cells and particles.
Conventionally, a method of purifying nucleic acids using a solid phase is known. For example, U.S. Pat. No. 5,234,809 discloses a method of purifying nucleic acids using a nucleic acid binding solid phase. Specifically, the method includes mixing a starting material, a chaotropic material and a nucleic acid binding solid phase, separating the solid phase with the nucleic acid bound thereto from the liquid, and washing the solid phase nucleic acid complexes.
However, this method is time consuming and complicated, and thus is not suitable for LOC. The method also has a problem of the use of the chaotropic material. That is, when the chaotropic material is not used, nucleic acids are not bound to the solid phase. The chaotropic material is harmful to humans, and thus should be handled with caution. Also, the chaotropic material acts as a material disturbing the subsequent step, and thus should be removed from purified nucleic acids during or after purification.
For the purpose of LOC implementation, the entire step of DNA isolation should be reduced and it is necessary to perform the PCR directly after cell lysis. To perform the PCR directly after cell lysis, the concentration of the resulting PCR inhibitors after cell lysis should be reduced. Thus, a method of removing the PCR inhibitors while performing cell lysis for the purpose of LOC implementation is required.
Thus, the inventors of the present invention researched for a method to overcome the above problems and discovered that nucleic acids can be effectively purified when cells or viruses are disrupted by vibrating a solution containing them using micro magnetic beads and a laser, the resulting PCR inhibitors attach to the magnetic beads, and then the magnetic beads to which the PCR inhibitors are attached are removed with a magnet attached to a capillary-shaped container wall.